1. Field of the Invention
The present invention is directed to methods of using halogenated peptides as internal standards for liquid chromatography-mass spectrometry and novel halogenated peptides useful for the same.
2. Description of the Related Art
As proteomics and systems biology converge, the need for the generation of high quality, large scale quantitative proteomic datasets has grown, and so-called label-free quantification has emerged as a very useful platform for their generation. Label-free quantitative experiments are usually designed to detect differentially abundant features in biologically relevant samples by comparing mass versus retention time feature maps generated by liquid chromatography mass spectrometry (LC/MS). Even though label-free proteomic experiments are time and cost effective, they require high levels of reproducibility at every step of the process. Too much variation resulting from sample preparation, liquid chromatography (LC) performance (e.g., injection, gradient delivery, flow rate), and MS performance (e.g., ionization efficiency, mass accuracy, detector performance) can lead to an increase in the false discovery rate (FDR) of detected peptides. Thus it is crucial to minimize such variation in order to adequately control the quality of the data. In addition, label-free experiments are often followed by directed MS/MS analyses, in which selected peptides are specifically targeted for identification, a procedure that also requires high system reproducibility. The total variation in the acquired data is the result of accumulating variation at each step. This variation, regardless of its source, be it from sample handling, injection irreproducibility, change in analyte volume, matrix and co-eluter interference (both suppression and enhancement), system instability, or finally variations in the ion source performance, can be accounted for if an appropriate internal standard system is used.
A more recent development in the field of quantitative proteomics is multi-reaction monitoring (MRM), also referred to as selected reaction monitoring (SRM). This MS-based technology is aimed at fast, sensitive and reproducible screening of large sets of known targets and is ideal for building biological assays in which the presence and quantity of specific analytes is being determined in multiple samples. Certain inputs, such as transitional values (m/z values for the precursor ion and its fragment ions), collision energies and chromatographic retention time are required to build a validated SRM/MRM assay. These values are either extracted from MS/MS data acquired from biological samples with the same type of instrument used for the SRM/MRM analyses, or from a set of peptide standards. To maximize the number of S/MRM measurements in one LC/MS/MS run, the use of elution time constraints has proven highly beneficial. Internal standards can therefore play an integral role in building S/MRM assays, if used to synchronize input values such as retention times between instruments or to monitor the retention time consistency in sequences of scheduled S/MRM experiments.
Internal standards are usually designed to best fit the analytical system for which they are being used. Since the currency of quantitative proteomics is ionized peptide ions, peptides thus represent the best candidates for internal standards for proteomic measurements. However, the use of peptides as internal standards can be challenging when trying to confidently detect the internal standard peptides in ion chromatograms acquired by mass spectral analysis of biological fluids, or other samples of similar complexity, where densely packed features cover the entire mass and time range. In addition, there is always a chance that a peptide with the same elemental composition as the internal standard might exist in the analyte, and thus completely throw off the calibration curve. The same argument is valid for heavy isotope labeled peptides since in many quantitative applications, the analytical matrix is made of heavy isotope labeled peptides. Hence, for a peptide to be useful as an internal standard in proteomic studies, it should have unique properties that make it easily detectable in a background of biological peptides.
Accordingly, while progress has been made in this field, there remains a need in the art for improved internal standards for liquid chromatography-mass spectrometry. The present invention fulfills these needs and provides further related advantages.